1. Field of the Invention
This invention relates to the field of high throughput solubility testing of compounds in flow cell devices. The invention relates in particular to the automated analysis of data collection and sample selection, as well as to automated procedures for realignment of sample flow.
2. Background
Solubility testing of new chemical entities (NCEs) is an important step in assessing their potential utility as pharmaceutical agents. Many compounds are rejected as having too low an aqueous solubility for adequate bioavailability to be useful in drug development.
Turbidity measurement has become popular as an indicator of the aqueous solubility of potential lead compounds. In drug development, turbidity is most commonly evaluated using microtiter plate-based nephelometers that measure the light scattering by the sample, or alternatively, standard laboratory turbidimeters which measure the change in transmitted light. In either case, compound precipitate is detected by passing light from a light source through some portion of the sample and evaluating light scattering.
With nephelometry plate readers, the sample is stationary (in the microtiter well), light is directed through the plate and off-axis forward-scattered light is used to detect the precipitate. By contrast, with turbidimeters, usually a portion of a stirred sample (often several milliliters) is passed through the turbidimeter and the reduction in transmitted light due to scattering is measured to obtain a turbidity reading.
At low concentrations of particulates, the change in transmitted light, viewed from straight on, is so slight that the reduction is virtually undetectable by any means. At higher concentrations the attenuation in transmitted light becomes easier to detect due to multiple scattering which interferes with direct transmission.
A solution to the problems described above is to measure the light scattered at an angle to the incident light beam and then relate this off-axis scattered light to the turbidity of the sample. Most instruments of this type measure the 90 degree scatter because light scattered at this angle is considered to correlate more precisely with particle concentration. These types of instruments are referred to as nephelometers.
Both of these technologies work by detecting multiple compound particles suspended within a given sample volume. The number of particles suspended in the liquid, the size of each particle and the light scattering properties of the particles are all critical factors that affect the sensitivity of a turbidimeter or a nephelometer. In addition, the intensity and focus of the light source and the sensitivity of the light detection mechanism are also important instrument properties that affect accuracy and sensitivity depending on the method used.
Nephelometry has been used extensively in the art to determine solubility limits of test compounds. See for example Dressman et al., Pharmaceutical Research, 15(1): 11-22 (1998). This technique has been adapted to high throughput screening by the capacity to read directly from microtiter plates. See for example Bevan and Lloyd, Analytical Chemistry 72: 1781-1787 (2000).
Light scattering has also been used for solubility testing of compounds for drug development (Lipinski et al. Advanced Drug Delivery Reviews 23: 3-25 (1997). These authors followed the absorbance increase due to light scattering by precipitated particulate material with a dedicated diode array UV machine.
Recently, the field has turned to high throughput flow cytometer systems adapted to light scattering to follow the precipitated particulates in a sample. See for instance Goodwin et al., A New Rapid Technique for Sensitive Solubility Measurements: A Flow Cytometric Approach. Published presentations from meetings of The Society for Biomolecular Screening, Edinburgh, Scotland, September 1999; and AAPS, New Orleans, La., November 1999.
Despite the successful application of flow cytometry to solubility testing, some problems still remain. Many problems associated with turbidimeters and nephelometers also plague the flow cell systems currently in use. For instance, non-uniformity of samples, due to impurities, solvent absorption, and precipitation of contaminants cause spurious light scattering. Also, anomalous scattering may be due to solvent scatter, dissolved impurities and such contaminants as solvent extractables from labware, e.g. microtiter plates etc.
There is still a need for an improved, accurate, robust flow cells system that is adaptable to high throughput screening of large numbers of compounds, that discriminates precipitated compound particles on the basis of size distribution from random background interference due to impurities from solvents, solvent extractables and other impurities and contamination. Further, the system should be capable of automatic detection and correction of misalignment of the sample liquid stream.
The present invention provides a flow cell system for solubility testing of a compound in a sample liquid; the system including the following components:
(a) a flow cell suitable for channeling a fluid sheath flowing through the flow cell, the fluid sheath directing a continuous or intermittent sample liquid stream, wherein the sample liquid stream comprises particles of the compound for solubility testing;
(b) a light source for illuminating the sample liquid stream in the flow cell and producing scattered light flashes from the particles of the compound;
(c) a detector for detecting the scattered light flashes and generating an electronic pulse detection signal for each light flash to provide raw sample data, each light flash having a light intensity and each electronic pulse detection signal having a pulse amplitude corresponding to the intensity of the detected light flash; and
(d) a means for real-time processing of multiple electronic pulse detection signals, wherein each of the signals is allocated to one of a series of channels, each channel detecting electronic pulse detection signals within a preset signal amplitude range, to provide multichannel sample data, and outputting the sample data for effectuating selection of a subsequent sample liquid from a plurality of subsequent sample liquids for solubility testing.
The flow cell according to the present invention may further include a means for storage and recall of the raw sample data and/or the multichannel sample data. Samples for solubility testing may be provided in the wells of a microtiter plate, which may include several dilutions of the sample for solubility testing.
The flow cell system of the present invention may also include a reservoir for containing a control substance for assessing the alignment of the sample liquid flow in the flow cell. The control substance may be a particle or a bead.
The invention also provides a method for solubility testing of a compound in a sample liquid; the method including the following steps:
(a) providing a flow cell suitable for channeling a fluid sheath flow through the flow cell, the fluid sheath flow directing a continuous or intermittent sample liquid stream, wherein the sample liquid stream comprises particles of the compound for solubility testing;
(b) illuminating the sample liquid stream in the flow cell to provide scattered light flashes from the particles of the compound;
(c) detecting the scattered light flashes to provide raw sample data and generating an electronic pulse detection signal for each light flash, each light flash having a light intensity and each electronic pulse detection signal having a pulse amplitude corresponding to the intensity of the detected light flash;
(d) processing of multiple electronic pulse detection signals in real-time, wherein each of the signals is allocated to one of a series of channels, each channel detecting electronic pulse detection signals within a preset signal amplitude range; and
(e) outputting the sample data to a means for effectuating a selection of a subsequent sample liquid from a plurality of subsequent sample liquids for solubility testing.
Multiple sample liquids for solubility testing may be provided in the wells of a microtiter plate. After solubility testing of the first sample liquid, subsequent sample liquids may include a series of higher dilutions of the same compound for solubility testing and may also optionally include a series of dilutions of one or more additional compounds for solubility testing.
The method of the present invention may be applied to a flow cell system that includes a reservoir for containing a control substance for assessing the alignment of the sample liquid flow in the flow cell. Alternatively, the control substance may be provided in one or more wells of a microtitier plate. The control substance employed may be a particle or a bead.
In another aspect the above methods further include the following steps for selection of the subsequent sample liquid: Either selecting one of the series of higher concentrations of the compound in the event that the multichannel sample data indicates the absence or low levels of particles of the compound; or selecting one of the series of increasing concentrations of the second compound for solubility testing in the event that the multichannel sample data indicates the presence of significant levels of particles of the compound.
The present invention also provides a means for real-time processing of multiple electronic pulse detection signals from a flow cytometer adapted for solubility testing, wherein each of the signals is allocated to one of a series of channels, each channel detecting electronic pulse detection signals within a preset signal amplitude range, to provide multichannel sample data, and outputting the sample data for effectuating selection of a subsequent sample liquid from a plurality of subsequent sample liquids for solubility testing.
The present invention further provides a means for real-time processing of multiple electronic pulse detection signals from a flow cytometer adapted for solubility testing, wherein each of the signals is allocated to one of a series of channels, each channel detecting electronic pulse detection signals within a preset signal amplitude range to provide multichannel control substance data, and thereby determining whether the sample liquid stream is aligned or misaligned, and outputting an effectuator signal if the sample liquid stream is misaligned, and initiating a corrective realignment process upon receiving the effectuator signal.